Method for manufacture of ceftiofur

ABSTRACT

A process for preparation of ceftiofur of formula (I) 
                         
having purity greater than 97% is disclosed. The process comprises reacting [2-(2-aminothiazol-4-yl)]-2-syn-methoxyimino acetic acid-2-benzothiazolyl thioester of formula (II),
 
                         
with 7-amino-3-(2-furanylcarbonylthiomethyl)-3-cephem-4-carboxylic acid of formula (III)
 
                         
in the presence of a mixture of an water-immiscible inert organic solvent and water and in the presence of a organic base and isolating ceftiofur of formula (I) substantially free of impurities by,
         a) adding water to the reaction mixture and selectively partitioning the impurities in the organic phase and ceftiofur (I) in the form of a salt with the base in the aqueous phase,   b) acidifying the aqueous phase containing ceftiofur (I) in the form of a salt with the base in the presence of a mixture containing a water-miscible and a water-immiscible organic solvent and in the presence of a saturated aqueous solution of an alkali or alkaline earth containing salt, to partition ceftiofur (I) in the organic phase, and   c) isolating ceftiofur (I) of high purity and substantially free of impurities by evaporation of the organic solvent or precipitation by addition of a anti-solvent.

FIELD OF THE INVENTION

The present invention relates to an improved method for manufacture ofCeftiofur in high purity. In particular, the present invention relatesto an improved method for manufacture of Ceftiofur substantially freefrom impurities.

BACKGROUND OF THE INVENTION

Ceftiofur is a broad-spectrum third generation antibiotic, which isprimarily used for veterinary use. It is known chemically as (6R,7R)-7-[[(2Z)-(2-amino-4-thiazolyl)(methoxyimino)acetyl]amino]-3-[[(2-furanylcarbonyl)thio]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid and is represented by the formula (I).

Ceftiofur is commercially sold as the sodium salt and is marketed underthe brand names Naxcel® and Excenel® for parenteral administration, inbovine animals.

Ceftiofur has been synthesized by any of the following three methods,viz,

I. Ceftiofur and its salts thereof, especially the sodium salt isdescribed in U.S. Pat. No. 4,464,367 (Labeeuw et. al). The patentdescribes two methods for preparation of Ceftiofur (I) comprising,

-   i) amidification at the 7-position of 7-amino-3-thiomethyl    furoyl-3-cephem-4-carboxylic acid with a suitably activated    [(2Z)-(2-tritylamino-4-thiazolyl) methoxyimino] acetic acid    derivative such as mixed anhydride or an activated ester to give    ceftiofur (I) after necessary deprotection.-   ii) functionalisation at the 3-position of a    7-[2-(2-aminothiazol-4-yl)-2-syn-methoxyimino] acetamido    cephalosporanic acid i.e. cefotaxime acid, with thiofuroic acid to    give Ceftiofur (I).

U.S. Pat. No. 4,464,367 contains enabling disclosure for preparation ofceftiofur as per method (I) comprising reaction of 7-amino-3-thiomethylfuroyl-3-cephem-4-carboxylic acid with the syn isomer of(2-tritylaminothiazol-4-yl)-2-methoxyimino acetic acid activated with1-hydroxy benzotriazole, followed by removal of the trityl protectinggroup to give ceftiofur (I).

However, for method (II), apart from the reaction sequence given incolumn 3, lines 25-35 depicting conversion of cefotaxime to ceftiofur(I), there is no enabling disclosure whatsoever about how the conversioncould be carried out.

The two methods for preparation of ceftiofur of formula (I) aresummarized in scheme (I).

Further, synthesis of ceftiofur as per method-I involves additionalsteps of protection of the amino group of[2-(2-aminothiazol-4-yl)]-2-syn methoxyimino acetic acid with tritylchloride followed by deprotection in presence of formic acid to giveceftiofur of formula (I). The amidification method utilises toxic,expensive dicyclohexyl carbodiimide for preparing the activated ester of[2-(2-aminothiazol-4-yl)]-2-syn methoxyimino acetic acid with 1-hydroxybenzotriazole, resulting in the formation of dicyclohexyl urea, which isdifficult to remove.

II. Although, the claims of U.S. Pat. No. 6,458,949 B1 (Handa, et al)appear to be obvious, over prior art described in EP Patent No.0,302,94, U.S. Pat. Nos. 5,109,135, and 5,109,131 the said patenthowever discloses another method for the preparation of Ceftiofur (I),which is summarized in scheme-II.

The method comprises, reaction of 4-halo-3-oxo-2-methoxyimino butyricacid, activated as the acid halide, with silylated 7-amino-3-thiomethylfuroyl-3-cephem-4-carboxylic acid to give the corresponding7-carboxamido derivative which on subsequent treatment with thioureagives ceftiofur of formula (I). Herein, the thiazole ring is formedafter the amidification step with thiourea.

The method described in U.S. Pat. No. 6,458,949 B1 is complicated andtedious, as it involves initial preparation of4-halo-3-oxo-2-methoxyimino butyric acid requiring four steps followedby subsequent amidification reaction with7-amino-3-thiofuroylmethyl-3-cephalosporanic acid and cyclisation withthiourea. The overall yield of (I) is therefore low, rendering themethod commercially not very attractive.

III. U.S. Pat. No. 6,388,070 (Deshpande, et. al) teaches a method forsynthesis of ceftiofur (I), and other cephalosporin compounds likeceftriaxone and ceftazidime, cefixime, cefpodoxime acid, cefetamet andcefotaxime acid.

Herein, [2-(2-aminothiazol-4-yl)]-2-syn methoxyimino acetic-acid isactivated as the thioester by reaction with2-mercapto-5-phenyl-1,3,4-oxadiazole in the presence ofbis-(2-oxo-oxazidinyl)phosphinic chloride. The thioester on reactionwith silylated 7-amino-3-substituted-3-cephem compounds in the presenceof a base at low temperatures gives the corresponding7-acylamido-3-substituted-3-cephem cephalosporin, depicted inscheme-III, herein below:

The activation of [2-(2-aminothiazol-4-yl)]-2-syn methoxyimino aceticacid with 2-mercapto-5-phenyl-1,3,4-oxadiazole is quite slow and itrequires a time upto 34 hours. Moreover, the method utilizes a reagentlike bis-(2-oxo-oxazidinyl)phosphinic chloride which is expensive.

Apart from the above methods, which utilize activation of the carboxylicacid moiety of either the [2-(2-aminothiazol-4-yl)]-2-syn methoxyiminoacetic acid or 4-halo-3-oxo-2-methoxyimino butyric acid through theformation of a mixed anhydride or activated ester with 1-hydroxybenzotriazole or 2-mercapto-5-phenyl-1,3,4-oxadiazole or an acidchloride, there are no reports of synthesis of ceftiofur (I) by othermethods, specially through utilization of other reactive derivatives ofthe said carboxylic compounds.

Various reactive derivatives of [2-(2-aminothiazol-4-yl)]-2-oxyiminoacetic acid compounds have been utilized for synthesis of several3-substituted cephalosporin antibiotics carrying a[2-(2-aminothiazol-4-yl)]-2-oxyimino acetamido addendum in the7-position. These include, to a name a few

-   a) an acyloxypliosphonium halide derivative as disclosed in U.S.    Pat. No. 5,317,099 (Lee et. al) for synthesis of cefotaxime and    ceftriaxone;-   b) an acetyl sulfite dialkyl formiminium halide hydrohalide    derivative as disclosed in U.S. Pat. No. 5,037,988 (Meseguer et. al)    for synthesis of cefotaxime, ceftriaxone, cefmenoxime, ceftizoxime,    and ceftazidime;-   c) a dialkyl chloro thiophosphate ester as disclosed in U.S. Pat.    No. 5,567,813 (Sung et. al) for synthesis of cefotaxime,    ceftriaxone, cefemenoxime, ceftizoxime, cefpirome sulfate and    cefepime;-   d) a dimethyl forminium chloride chlorosulphate derivative as    disclosed in U.S. Pat. No. 5,739,346 (Datta et. al) for synthesis of    cefotaxime, ceftriaxone, ceftazidime, cefazolin etc.

A reactive derivative of [(2)-(2-aminothiazol-4-yl)]-2-syn-oxyiminoacetic acid compounds widely utilized in cephalosporin chemistry foreffecting amidification at 7-position is the 2-benzothiazolyl thioester,first disclosed in U.S. Pat. No. 4,767,852 (Ascher et. al), thechemistry of which is shown hereinbelow.

wherein the groups R¹ to R³ are as defined therein and the group R⁴ isan acetoxy, carbamoyloxy, or is a group of formula S—Y, wherein Y is aheterocyclic ring.

The cephalosporin compounds that fall under the definition of the groupR⁴ of this patent include the commercially and therapeutically valuablecephalosporin antibiotics like cefotaxime, ceftriaxone and cefuzonam.

The group R⁴ of U.S. Pat. No. 4,767,852 does not encompass ceftiofur asit covers only those 3-thiomethyl compounds in which the sulphur atom isdirectly attached to a heterocyclic ring, and not those compounds inwhich a carbonyl group is interposed between S and Y, where Y is aheterocyclic ring Ceftiofur (I) has a carbonyl group interposed betweenS and Y i.e. between the 3-thiomethyl group and the furan ring.

The method disclosed in U.S. Pat. No. 4,767,852, as is evident fromdescription given in the examples of the said patent, essentiallyconsists reaction of a protected 7-amino-3-substituted cephalosporanicacid derivative, in particular a (N,O)-bis silylated7-amino-3-substituted cephalosporanic acid derivative with the[2-(2-aminothiazol-4-yl)]-2-syn-oxyimino acetic acid-2-benzothiazolylthioester in an inert organic solvent at ambient temperature for a timeranging between 0.5 to 48.0 hours to give the object[2-(2-aminothiazol-4-yl)]-2-syn-oxyiminoacetamido-3-substituted-3-cephem-4-carboxylic acid compounds such ascefotaxime, ceftriaxone and cefuzonam, which it should be noted carries“a residue of a nucleophile at the 3α-position”.

From the abovementioned methods it is apparent that the preparation ofthe [2-(2-aminothiazol-4-yl)]-2-oxyiminoacetamido-3-substituted-3-cephem4-carboxylic acid compounds is effectedessentially in an inert organic solvent, thereby implying a reactionmedium free of water and through utilization of a protected7-amino-3-substituted cephalosporanic acid derivative as the startingmaterial.

In addition, the [2-(2-aminothiazol-4-yl)]-2-oxyimino aceticacid-2-benzothiazolyl thioester has also been utilized for synthesis ofother [2-(2-aminothiazol-4-yl)]-2-oxyiminoacetamido-3-substituted-3-cephem-4-carboxylic acid compounds such ascefixime as disclosed in U.S. Pat. No. 6,313,289. Here again, the methodcomprises reacting a protected 7-amino-3-substituted cephalosporanicacid derivative, particularly a protected7-amino-3-vinyl-3-cephem-4-carboxylic acid, wherein the amino group andthe carboxylic group are protected as trialkylsilyl group with a[2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid-2-benzothiazolylthioester in the form of a aqueous solution of a crystalline solvatewith dimethyl acetamide and an inert organic solvent medium at roomtemperature in the presence of a base to give cefixime after removal ofthe said protective groups.

Cefixime is isolated as the salt of an organic base or as an acidaddition salt with sulphuric acid.

In their attempt to extend the method described in U.S. Pat. Nos.4,767,852 and 6,313,289, for synthesis of ceftiofur the presentinventors found to their surprise that when (N,O)-bissilyl-7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula(III¹),

is reacted with [2-(2-aminothiazol-4-yl)]-2-methoxyimino aceticacid-2-benzothiazolyl thioester (MAEM) of formula (II),

in an inert organic solvent (dichloromethane) in the presence of anorganic base (triethyl amine) at ambient temperature (15-30° C.), themethods had the following disadvantages, which are undesirable for anycommercial process. These are,

-   i) the reaction required a period of more than 20-24 hours to    proceed to completion,-   ii) the conversion to ceftiofur as indicated by monitoring of the    reaction by HPLC was only about 88-90%,-   iii) about 9-10% of impurities were formed in the reaction,-   iv) the product i.e. ceftiofur was isolated as a gummy material, and-   v) the isolated product i.e. ceftiofur had a purity of only about    88% containing about 10-12% of impurites. Purification of the    material thus obtained resulted in considerable loss thus giving    ceftiofur (I) in low yield.

An improved method for synthesis of ceftriaxone comprising reaction of[2-(2-aminothiazol-4-yl)]-2-syn-methoxyimino aceticacid-2-benzothiazolyl thioester of formula (III) with a unprotected7-amino-3-substituted cephalosporanic acid derivative i.e.7-amino-3-(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-1,2,4-triazin-3-yl)thiomethyl-3-cephem-4-carboxylicacid has been disclosed in U.S. Pat. No. 5,026,843 (Riccardo et. al.).The improvement comprises carrying out the said amidification reactionin a monophasic system comprising a mixture of a water-miscible organicsolvent and water and in the presence of an organic base. Theceftriaxone thus obtained without isolation is converted into its sodiumsalt, which is isolated from the reaction mixture. The chemistry issummarized hereinbelow:

The water-miscible organic solvents listed in the U.S. Pat. No.5,026,843 as useful for carrying out the abovementioned synthesisinclude tetrahydrofuran, dimethyl acetamide, dimethyl formamide,dioxane, dimethoxyethane. When these solvents are mixed with water, ahomogeneous single phase would result. This helps in keeping theceftiaxone thus produced in solution throughout the reaction andthereby, enabling a one-pot reaction for preparation of ceftriaxonesodium.

An attempt by the present inventors to extend the method described inU.S. Pat. No. 5,026,843 for synthesis of ceftiofur or ceftiofur sodiumcomprising reaction of [2-(2-aminothiazol-4-yl)]-2-methoxyimino aceticacid-2-benzothiazolyl thioester (MAEM) of formula (II) with7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula (III),

in a medium consisting of water and a water-miscible organic solventdisclosed in the said patent like tetrahydrofuran andN,N-dimethylacetamide, was however, not satisfactory and was found togive the product i.e. ceftiofur (I) associated with impurities in thelevel of 5-10% depending on the water-miscible organic solvent used. Theproduct obtained was a sticky solid adhering to the sides of thereaction vessel, rendering its isolation as a solid very difficult.

In addition, replication of the methods exactly as described in U.S.Pat. Nos. 4,464,367 and 6,458,949 B1, referred herein earlier were alsofound to be associated with formation of higher level of impurities,which are to the tune of about 25-28% and 5-10% respectively.

The level of impurites formed in the-synthesis of ceftiofur by the fourmethods discussed herein before are summarized in Table-I.

TABLE I The level of impurities formed in the synthesis of ceftiofur (I)by various methods Total % Impurities No. Method Conversion (%) % YieldPurity % 1 As per that described in 75 25-28 Gummy material — U.S. Pat.No. 4,464,367 (not isolated) 2 As per that described in 91.2 8.8 69.692.78 U.S. Pat. No. 6,458,949 B1 3 Extrapolation of the method 89 11Sticky solid — described in U.S. Pat. No. 4,767,852* (not isolated) 4Extrapolation of the method 93  5-10 Sticky solid — described in U.S.Pat. No. 5,026,843** (not isolated) *reaction of (N,O)-bissilyl-7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula(III¹) with [2-(2-aminothiazol-4-yl)]-2-methoxyimino aceticacid-2-benzothiazolyl thioester of formula (II) in dichloromethane atambient temperature **reaction of7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula (II) with[2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid-2-benzothiazolylthioester of formula (III) in a medium containing water and awater-miscible organic solvent in presence of a base

It might be mentioned herein that the 3-thiofuroylmethyl substituent inceftiofur, by virtue of it containing a carbonyl group interposedbetween a sulfur atom and a furan ring system is very labile in natureand is highly susceptible to fission of the sulfur-carbonyl bond as wellas highly prone to undergo dimerisation, leading to formation of adimeric compound in solution. This sets apart ceftiofur (I) from other3-heterocyclic thiomethyl cephalosporin derivatives as described in U.S.Pat. No. 4,767,852 wherein the abovementioned lability is lesspronounced or negligent and thereby ensuring their synthesis through anyknown methods.

The structure of the impurities arising out of fission of thesulfur-carbonyl bond and dimerization are given herein below ascompounds (IV) and (V) respectively.

Thus, to summarize,

-   i) Synthesis of ceftiofur (I) had been achieved in prior art either    through amidification at the 7-amino position of (a suitably    protected) 7-amino-3-thiofuroylmethyl-3-cephalosporanic acid with    [2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid activated    through formation of a mixed anhydride or through formation of an    activated ester with 1-hydroxybenzotriazole, or    2-mercapto-5-phenyl-1,3,4-oxadiazole and described respectively in    U.S. Pat. Nos. 4,464,367 and 6,388,070 B1, summarized in Scheme-I    and Scheme-III, or through amidification at the 7-amino position of    (a suitably protected) 4-halo-2-methoxyimino-3-oxo butyric acid    activated as the acid halide, followed by cyclization of the    intermediate compound thus formed with thiourea, as described in    U.S. Pat. No. 6,458,949 B1 and summarized in Scheme-II;-   ii) Functionalization at 3-position of cefotaxime by reaction with    thiofuroic acid, as summarized in Scheme-I, for the conversion of    which, however, there is no enabling disclosure whatsoever in U.S.    Pat. No. 4,464,367;-   iii) Replication of both the abovementioned methods was found to    give ceftiofur associated with impurities in the range of 5-28%;-   iv) Extension of the methods described in U.S. Pat. Nos. 4,767,852    and 5,026,843 for synthesis of ceftiofur (I) was not only lengthy    requiring about 18-24 hours but also lead to higher levels of    impurity, resulting in a gummy material.-   v) The abovementioned methods involve protection and deprotection of    reactive functional groups, increasing the cost and time of    manufacture;-   vi) The product i.e. ceftiofur obtained by the abovementioned    methods, because of the higher level of impurities is therefore not    suitable for formulation into a suitable dosage form; and-   vii) Removal of the impurities by purification leads to considerable    loss of the product, increasing the cost of manufacture and    rendering such methods commercially not attractive.

Further, since regulatory authorities all over the world are highlyconcerned about the level of impurities in a drug substance/drug productand are becoming increasingly stringent in approving products containinglevels of impurities above the prescribed limits for human or animalconsumption, it is imperative that any method of manufacture of a drugsubstance/drug product, apart from being commercially viable shouldprovide the product conforming to pharmacopoeial specifications,containing amount of impurities within the prescribed limits orsubatantially free of such impurities.

In view of the foregoing reasons, there exists a need for a vastlyimproved method for manufacture of ceftiofur (I), which not onlysatisfies the techno-commercial aspects i.e. cost-effectiveness, ease ofoperations, etc, but also provides a product of high purity, free ofimpurities and possess properties, which are amenable for formulationinto a suitable dosage form.

The present inventors have found to their surprise that the existingneed for an improved method for manufacture of ceftiofur in high yieldand high purity could be achieved through:

-   i) carrying out amidifaction at 7-amino position of    7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula (III)    with [2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid activated    as its -2-benzothiazolyl ester of formula (II), obviating the need    for protection and deprotection of reactive functional groups,-   ii) the said amidification reaction is carried out in a system    comprising mixture of a water-immiscible inert organic solvent and    water i.e. a biphasic system and in the presence of a base, in a    remarkably shorter time (1.5 to 3.0 hours) resulting in a product    with substantially lower level of impurities,-   iii) removal of most of the impurities formed during the above    reaction in (ii) above, through a selective extraction method to    provide ceftiofur (I) of high purity and substantially free of    impurities by extraction of the aqueous mixture of the alkyl    ammonium salt of ceftiofur (I) with a inert organic solvent.-   iv) further removal of impurities formed during acidification of the    alkyl ammonium salt of ceftiofur (I) with a mineral acid, in the    presence of a mixture of water-miscible and water-immiscible solvent    and in the presence of a saturated aqueous solution of an alkali or    alkaline earth containing salt, by selectively partitioning    ceftiofur (I) in the organic phase substantially free from    impurities with the associated impurities getting extracted in the    aqueous phase.

OBJECTS OF THE INVENTION

An object of the invention is to provide a method for manufacture ofceftiofur of formula (I) of high purity and substantially free ofimpurities.

Another object of the present invention is to provide a method formanufacture of ceftiofur of formula (I) comprising reaction of7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula (III) with[2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid activated as its-2-benzothiazolyl ester of formula (II) in a biphasic system comprisingmixture of a water-immiscible inert organic solvent and water and in thepresence of base, wherein the formation of impurities is substantiallyminimized with concomitant higher conversion to the desired product.

Yet another object of the present invention is to provide a selectivemethod for isolation of ceftiofur of formula (I), providing the productin high yield and high purity and substantially free of impurities.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a simple, cost-effectivemethod for manufacture of ceftiofur of formula (I),

comprising reaction of [2-(2-aminothiazol-4-yl)]-2-methoxyimino aceticacid-2-benzothiazolyl ester of formula (II), and

7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula (III),

in the presence of a mixture comprising a water-immiscible inert organicsolvent, and water and in the presence of a base and isolation to giveceftiofur of formula (I), in high purity and substantially free ofimpurities.

In another aspect, the present invention provides a method for isolationof ceftiofur of formula (I), in high purity and substantially free ofimpurities comprising,

-   i) addition of water to the reaction mixture and selective    partitioning of the impurities in the organic phase and    ceftiofur (I) in the form of a salt with the base in the aqueous    phase,-   ii) acidification of the aqueous phase containing ceftiofur (I) in    the form of a salt with the base in the presence of a mixture    containing a water-miscible and a water-immiscible organic solvent    and in the presence of a saturated aqueous solution of air alkali or    alkaline earth containing salt, to partition ceftiofur (I) in the    organic phase, and-   iii) isolation of ceftiofur (I) of high purity and substantially    free of impurities by evaporation of the organic solvent or    precipitation by addition of a co-solvent.

The method for preparation of ceftiofur (I) as per the present inventionis summarized in Scheme-V for ready reference,

DETAILED DESCRIPTION OF THE INVENTION

The starting materials required are prepared by known methods.7-Amino-3-thiomethyl furoyl-3-cephem-4-carboxylic acid of formula (III)can be prepared by the method disclosed in U.S. Pat. No. 4,937,330;comprising reaction of thiofuroic acid of formula (VI) with 7-aminocephalosporanic acid of formula (VII) at pH 6.4 and temperature of 65°C. in a mixture of water and an inert solvent such as ethyl acetate.

[2-(2-aminothiazol-4-yl)]-2-syn-methoxyimino aceticacid-2-benzothiazolyl thioester (MAEM) (II) can be prepared by themethod described in EP Patent No. 0,037,380 comprising reaction of[2-(2-aminothiazol-4-yl)-2-syn-methoxyimino]acetic acid (IX) withbis[benzthiazolyl-(2)]disulphide (X) in the presence of triphenylphosphine and dichloromethane as solvent at 0° C. The product (II)separating out is filtered, washed with dichloromethane followed bystirring with ethyl acetate at 0° C. and filtration to give[2-(2-aminothiazol-4-yl)]-2-syn-methoxyimino aceticacid-2-beizothiazolyl thioester of formula (II).

In a typical method for preparation of ceftiofur of formula (I),7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula (III) isadded to a mixture of the water-immiscible inert organic solvent andwater, followed by addition of the base. To the mixture is added[2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid-2-benzothiazolylester of formula (II) and the reaction mixture agitated till completionof reaction to give ceftiofur of formula (I).

By definition an inert water-immiscible organic solvent is one, whichdoes not participate in the reaction but helps facilitate smoothconversion of the reactants into the end product i.e. ceftiofur.

Typical of such water-immiscible inert organic solvents that can beemployed are selected from chlorinated hydrocarbons such asdichloromethane, dichloroethane, chloroform etc

While, any polar solvent, say a C₁₋₄ alkanol can be used instead ofwater for conducting the reaction in admixture with the water-immiscibleinert organic solvent, resulting in a homogeneous phase, such polarsolvents, however, do not contribute in reducing the formation ofimpurities. Water, on the other hand, when employed in admixture with awater-immiscible inert organic solvent ensures a heterogenous biphasicsystem, which substantially helps minimization of the impurities.

The addition of water helps in partial dissolution of the reactants as aresult of which agitation of the reaction mass becomes easier. Asbenceof water in the reaction medium makes agitation of the reaction massvery difficult.

The high selectivity of water over a C₁₋₄ alkanol in affecting theminimization of impurities can be understood form Table-II.

Table-II: The effect of water and a C₁₋₄ alkanol (methanol) in admixturewith a water-immiscible inert organic solvent in the level of impuritiesformed in the method for preparation of ceftiofur (I) by reaction of7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula (III) with[2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid-2-benzothiazolylester of formula (II)

Impurities (%) Formed in the Present in the No. Reaction Medium reactionisolated product 1 Water + Dichloromethane 4.00 1.30 2 Methanol +Dichloromethane 11.00 4.00

While the ratio of the water-immiscible inert organic solvent to watercan vary from 90:10 of the former to the latter to 98:2.0 of the same,the best results are obtained when the ratio is between 95:5.0 to97.5:2.5 of the water-immiscible inert organic solvent to water.

For instance, the best results are obtained when the ratio ofwater-immiscible inert organic solvent to water is 97.5:2.5.

The dramatic effect brought about by the addition of water can berationalised as follows:

7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula (III) onaddition of an organic base is converted to its alkyl ammonium salt(III²), which is soluble in the aqueous phase. The alkyl ammonium salt(III²) reacts with the compound of formula (II), which is soluble in theinert water immiscible solvent, apparently at the interface between theaqueous and organic phase thereby minimizing/eliminating side reactionsby a mechanism which has not been clearly understood and facilitatinghigher conversion with lower impurity formation.

Interestingly enough, synthesis of ceftiofur of formula (I) has not beenachieved earlier utilising a heterogenous biphasic system.

The reaction can be carried out a temperature ranging between low toambient i.e. from 0° C. to 30° C., but preferably at a temperatureranging between 0° C. to 15° C.

Although any base can be used, organic bases are preferred. Typical ofsuch bases that can be employed include triethylamine, tri-n-butylamine,tert-butylamine, dicyclohexylamine, N-methyl morpholine,2,3-dimethylaminopyridine, N-methyl pyrrolidinone etc.

Among all the bases, tertiary amines are preferred which are selectedfrom triethyl amine, tri-n-propyl amine, tri-n-butyl amine. Triethylamine is preferred among the tertiary amines as impurity formation isminimized.

The rise in impurity formation by using organic bases having highernumber of carbon atoms may be presumably due to the increase in thehydrophobicity of the trialkyl ammonium salt (III²) with the increase inthe number of carbon atoms in the trialkyl amine. This increase inhydrophobicity leads to increase in the solubility of the trialkylammonium salt (III²) in the hydrophobic solvent due to which theacylation takes place in the hydrophobic solvent and not at theinterphase between the aqueous phase and the hydrophobic phase resultingin a rise in the level of impurities.

The base can be employed in molar proportions of 1.0 mole to 3.0 molesper mole of compound of formula (III). Preferably the base is employedin molar proportions of 1.0 moles to 2.0 moles per mole of the compoundof formula (III).

[2-(2-aminothiazol-4-yl)]-2-syn-methoxyimino aceticacid-2-benzothiazolyl thioester of formula (II) is employed in molarproportions of 1.0 to 2.0 moles per mole of the compound of formula(III), but preferably in a molar proportion of 1.0-1.5 moles.

The progress of the reaction can be monitored by TLC or HPLC andtypically depending upon the temperature employed the reaction is overin a period ranging from 1.0 to 3.0 hours, with the monitoring systemindicating more than 95% conversion to ceftiofur (I).

At the end of the reaction water is added, the aqueous layer isseparated and further washed with a water-immiscible organic solventselected from a chlorinated solvent such as dichloromethane,dichloroethane, chloroform or C₁₋₆ alkyl acetate like ethyl acetate,propyl acetate, n-butyl acetate at least once.

To the aqueous layer is added a mixture of a water-miscible solvent anda water-immiscible solvent, after separation of the organic layer.

The water-miscible organic solvent is selected from nitrites such asacetonitrile, propionitrile, butyronitrile or ketones such as acetone,methyl ethyl ketone or polar aprotic solvents like dimethyl sulphoxide,sulpholane. The preferred water-miscible solvent is a nitrile,preferably acetonitrile.

The water-immiscible solvent is selected from chlorinated solvents likedichloromethane, dichloroethane, chloroform or C₁₋₆ alkyl acetates likeethyl acetate, n-butyl acetate, isopropyl acetate but preferably ethylacetate.

A mixture of alkyl acetate preferably ethyl acetate and nitrilepreferably acetonitrile is added to the aqueous layer containing thealkyl ammonium salt of ceftiofur (I).

The ratio of the mixture of water-miscible solvent and water-immisciblesolvent is between (1:1) and (5:1) but preferably the ratio of alkylacetate and the polar aprotic solvent is (2:1 or 3:1).

The pH of the mixture is adjusted to 3.0±0.1 by addition oforthophosphoric acid and the mixture agitated further for completeliberating the ceftiofur (I) free acid from its corresponding salt witha organic base and taking it to the organic phase.

A mineral acid selected from hydrochloric acid, sulphuric acid,orthophosphoric acid but preferably orthophosphoric acid is added to thebiphasic mixture.

A saturated solution of an alkali or an alkaline earth metal containingsalt (15-30%) is added to the biphasic system and agitated at ambienttemperature. The organic layer is separated and the aqueous layer isoptionally again extracted with a mixture of water-miscible solvent anda water-immiscible solvent.

The alkali or an alkaline earth metal containing salt is selected fromsodium chloride, sodium sulphate, potassium chloride, potassiumsulphate, calcium chloride, but preferably sodium chloride.

Ceftiofur of formula (I) is isolated from the organic layer byevaporation of the organic solvent or by addition of a co-solvent.

The co-solvent added for precipitating out centiofur (I) at ambienttemperature from the organic layer can be a non-polar aromatic solventlike toluene, xylene or aliphatic solvent like cyclohexane, n-hexane,heptane.

The mixture is agitated between 45-90 minutes but preferably 60 minutesfor complete crystallization of pure ceftiofur (I). The pure compound(I) is filtered, washed with cyclohexane and dried at 35-40° C. Thepurity of ceftiofur (I) thus obtained has purity above 97% andsubstantially free from impurities such as the dimer compound (IV) andthiol compound (V).

Ceftiofur acid of formula (I) thus prepared, exhibits remarkablestability under stringent temperature and humidity conditions of 40±2°C. and relative humidity of 75%±5° C. even after 3 months of storage.

In a specific embodiment, 7-Amino-3-thiomethylfuroyl-3-cephem-4-carboxylic acid (1.0 mole) of formula (III) is addedto dichloromethane and the mixture cooled between 0 and 5° C. Triethylamine (1.8 moles) is added to the mixture followed by[2-(2-aminothiazol-4-yl)]-2-syn-methoxyimino aceticacid-2-benzothiazolyl thioester (1.2 moles) of formula (II) is added tothe mixture with agitation. Water [0.4 times volume per gram of thecompound of formula (III)] is added to the reaction mixture and agitatedbetween 1.5 to 3.0 hours for the reaction to go to completion. Thereaction mixture is quenched with water and the organic layer separated.The aqueous layer containing the salt of ceftiofur (I) is extracted withdichloromethane at least twice followed by extraction with ethyl acetateat least once.

A (2:1) mixture of ethyl acetate and acetonitrile is added to theaqueous layer and agitated at ambient temperature. The pH of the mixtureis adjusted to 3.0±0.1 with orthophosphoric acid (30%) at ambienttemperature. A 20% aqueous solution of sodium chloride is added to thebiphasic mixture and agitated. The organic layer is separated and afteroptional treatment with activated carbon followed by filtration throughcelite bed is concentrated to isolate ceftiofur of formula (I).Alternately, cyclohexane is added to the organic layer and ceftiofur offormula (I) is allowed to precipitate completely at ambient temperature.The mixture is filtered and the wet cake washed with cyclohexanefollowed by drying at 35-40° C. to give ceftiofur (I) substantially freefrom impurities and with purity greater than 97%.

A comparison of the present method with that of prior art methods forpreparation of ceftiofur (I) is given in Table-III.

TABLE III Synthesis of Ceftiofur (I) by the method of the presentinvention vis-à-vis the methods reported in prior art. Method of U.S.Pat. No. Method of U.S. Pat. No. Method as described Method as described4,767,852, extrapolated 5,026,843, extrapolated Method of Present inU.S. Pat. No. in U.S. Pat. No. for synthesis of for synthesis of No.Invention 4,464,367 6,458,949 ceftiofur (I). ceftiofur (I) 1. Steps Onestep. Requiring Two steps. Requiring Seven steps starting One stepstarting from One step starting from required no protection of theprotection and from preparation of 7-amino-3-substituted-7-amino-3-substituted- for making 2-amino group of deprotection of the4-halo-3-oxo-2- 3-cephem carboxylic 3-cephem carboxylic ceftiofur[2-(2-aminothiazol- 2-amino group of methoxyimino acid. acid. (I)4-yl)]-2- [2-(2-aminothiazol- butyric acid. synmethoxyimino 4-yl)]-2-acetic acid synmethoxyimino derivative. acetic acid derivative. 2.Method With [2-(2- With [2-(2- With 4-halo-3-oxo-2- With [2-(2- With[2-(2- for acylation aminothiazol- aminothiazol-4-yl)- methoxyiminobutyric aminothiazol-4-yl)-2- aminothiazol-4-yl)-2- of the 7-4-yl)-2-syn- 2-syn-methoxyimino] halide on silylated 7-syn-methoxyimino] syn-methoxyimino] amino group methoxyimino] aceticacid (IX) amino-3-[2-furyl acetic acid (IX) acetic acid (IX) acetic acidactivated as 1-Hydroxy carbonylthiomethyl]- as its 2- as its 2- (IX) asits 2- benzotriazole 3-ccphem-4- mercaptobenzothiazolemercaptobenzothiazole mercaptobenzothiazole derivative in presencecarboxylic acid ester ester ester. of dicyclohexyl carbodiimide. 3.Reaction Between 1.5 to Between 2.0 to Between 2.0 to Between 18 toBetween 2.0 to time 3.0 hours 4.0 hours 4.0 hours. 24 hours 4.0 hours 4.Level of 4.0 to 5.0% 26.0 to 29.0% 7.0 to 8.0% 8.0 to 10.0% 8.0 to 10.0%impurities 4. Level of 4.0 to 5.0% 26.0 to 29.0% 7.0 to 8.0% 8.0 to10.0% 8.0 to 10.0% impurities formed during the reaction 5. Level of 1.5to 3.0% ≈28.0% 7.0% — — impurities gummy solid gummy solid in isolated(not isolated) (not isolated) ceftiofur (I) 6. Purity of greater than97%. — between 90-95% — — ceftiofur (I) 7. Stability Stable for 3 monthsImpurity level is Starts degrading in — — of ceftiofur at 40 ± 2° C. andquite high, therefore, the first month at sodium relative humiditystability is low. 40 ± 2° C. and prepared 75% ± 5 relative humidity fromthe 75% ± 5; purity acid (I) falls below specified limits in the firstmonth itself.

Ceftiofur sodium can be prepared from ceftiofur acid (I) by methodsdisclosed in the prior art. Ceftiofur acid (I) prepared by the presentmethod has been converted to its sodium salt by a method disclosed in aco-pending application No. 938/MUM/2002 dated Oct. 29, 2002.

Ceftiofur sodium prepared from ceftiofur acid (I) made by the presentmethod has higher stability due to the lower level of impuritiesgenerated during the preparation of ceftiofur acid (I) and is alsoamenable to formulation as a dosage form.

The invention can be further illustrated by the following examples,which however should not be construed to be limiting the scope of theinvention.

EXAMPLE 1 Preparation of (6R,7R)-7-[[(2Z)-(2-amino-4-thiazolyl)(methoxyimino)acetyl]amino]-3-[[(2-furanylcarbonyl)thio]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid (I). (Mixture of Water Immiscible Solvent and Water in the Presenceof Triethyl Amine as Base)

7-amino-3-(2-furanylcarbonylthiomethyl)-3-cephem-4-carboxylic acid(III)(100 gms; 0.294 moles) was added to dichloromethane (1000 ml). Thereaction mixture was cooled to 0° C. and triethyl amine (53.57 gms;0.529 moles) was added at 0-5° C. in 60 minutes.[2-(2-aminothiazol-4-yl)]-2-syn-methoxyimino aceticacid-2-benzothiazolyl thioester (II) (123.5 gms; 0.353 moles) was addedand agitated for 15 minutes. Water (25.0 ml) was added to the mixtureand agitated at 5-7° C. The reaction was monitored by HPLC and themixture stirred till compound (III) was less than 1.0% on HPLC. Thereaction mixture was worked up by adding water (700 ml) and stirred for15 minutes at 10-15° C. The aqueous layer was separated and washedthrice with dichloromethane (300 ml). The aqueous layer was then againwashed with ethyl acetate (300 ml). The aqueous layer was separated anda mixture of ethyl acetate (2000 ml) and acetonitrile (1000 ml) wereadded to the aqueous layer. The pH was adjusted to 3.0 by adding 25%orthophosphoric acid in 30 minutes at 15-20° C. A concentrated solutionof sodium chloride (25%) was added to the biphasic mixture and theresulting biphasic mixture was agitated for 30 minutes. The organiclayer was separated and the aqueous layer re-extracted with a (2:1)mixture of ethyl acetate and acetonitrile (750 ml). The organic layerswere combined and washed with 5% brine solution. The organic layer wasseparated and after optional carbon treatment followed by filtration wasdried on sodium sulphate. The organic layer was evaporated and a mixtureof ethyl acetate (300 ml) and cyclohexane (1500 ml) was added to theresidue and agitated at 20-25° C. for 60 minutes. The product (I) wasfiltered and washed twice with cyclohexane (200 ml). The product wasdried at 35-40° C. under reduced pressure to give 100.3 gms of ceftiofur(I). % yield: 66.95; Purity: 98.5%. Total impurity: 1.50%

EXAMPLE 2 Preparation of (6R,7R)-7-[[(2Z)-(2-amino-4-thiazolyl)(methoxyimino)acetyl]amino]-3-[[(2-furanylcarbonyl)thio]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid (I) (Water Immiscible Solvent and Water Miscible Solvent WithoutWater)

7-amino-3-(2-furanylcarbonylthiomethyl)-3-cephem-4-carboxylic acid (III)(5.0 gms; 0.0147 moles) was added to dichloromethane (50 ml). Thereaction mixture was cooled to 0° C. and[2-(2-aminothiazol-4-yl)]-2-syn-methoxyimino aceticacid-2-benzothiazolyl thioester (II) (5.4 gms; 0.0154 moles) was addedto the mixture with agitation. Triethyl amine (2.67 gms; 0.0264 moles)was added to the mixture followed by methanol (2.5 ml). The reactionmixture was monitored by HPLC and stirred for 3.0 hours at 5±2° C.; thereaction was not going to completion as 2.5% was remaining unreactedeven after stirring further for 2.0 hours. The reaction mixture wasquenched with water. The aqueous layer was washed with dichloromethane(45 ml) at least once followed by washing with ethyl acetate (45 ml).Ethyl acetate (15 ml) was added to the aqueous layer and the pH wasadjusted to pH 3 with 30% orthophospl-ioric acid. The organic layer wasseparated dried on sodium sulphate and evaporated between 25-30° C.under reduced pressure. A mixture of ethyl acetate (300 ml) andcyclohexane (1500 ml) was added to the residue and agitated at 20-25° C.for 60 minutes. The product (I) was filtered and washed twice withcyclohexane (200 ml). The product was dried at 35-40° C. under reducedpressure to give 4.66 gms. % yield: 64.5%; Purity: 96.1%. Totalimpurity: 3.9%.

1. A process for preparation of ceftiofur of formula (I) of a puritygreater than 97%

comprising the steps of: reacting[2-(2-aminothiazol-4-yl)]-2-syn-methoxyimino aceticacid-2-benzothiazolyl thioester of formula (II),

with 7-amino-3-(2-furanylcarbonylthiomethyl)-3-cephem-4-carboxylic acidof formula (III)

in the presence of a mixture of an water-immiscible inert organicsolvent and water and in the presence of a organic base and isolatingceftiofur of formula (I) of a purity greater than 97% by, a) addingwater to the reaction mixture and selectively partitioning theimpurities in the organic phase and ceftiofur (I) in the form of a saltwith the base in the aqueous phase, b) acidifying the aqueous phasecontaining ceftiofur (I) in the form of a salt with the base in thepresence of a mixture containing a water-miscible and a water-immiscibleorganic solvent and in the presence of a saturated aqueous solution ofan alkali or alkaline earth containing salt, to partition ceftiofur (I)in the organic phase, and c) isolating ceftiofur (I) of a purity greaterthan 97% by evaporation of the organic solvent or precipitation byaddition of an anti-solvent.
 2. The process according to claim 1,wherein the water-immiscible inert organic solvent comprises achlorinated solvent.
 3. The process according to claim 2, wherein saidchlorinated solvent is selected from dichloromethane,1,2-dichloroethane, and chloroform.
 4. The process according to claim 1,wherein the organic base is selected from triethyl amine, N-methylmorpholine, tert-butyl amine, dicyclohexyl amine, tri-n-butylamine,N-methyl pyrrolidinone and 2,3-dimethylamino pyridine.
 5. The processaccording to claim 4, wherein the base is employed in molar proportionof 1.0 to 3.0 moles per mole of the compound of formula (III).
 6. Theprocess according to claim 1, wherein the compound of formula (II) isemployed in molar proportion of 1.0 to 2.0 moles per mole of thecompound of formula (III).
 7. The process according to claim 1, whereinthe ratio of the water-immiscible inert organic solvent to water isbetween 90:10 and 98:2.0.
 8. The process according to claim 7, whereinthe ratio of the water-immiscible inert organic solvent and to water isbetween 95:5.0 and 97.5:2.5.
 9. The process according to claim 1,wherein the temperature at which the reaction is carried out is between0 and 30° C.
 10. The process according to claim 1, wherein thewater-immiscible solvent is a chlorinated solvent or C₁₋₆ alkyl acetate.11. The process according to claim 10, wherein the chlorinated inertorganic solvent is selected from dichloromethane, dichloroethane andchloroform and the C₁₋₆ alkyl acetate is selected from ethyl acetate,butyl acetate, n-propyl acetate, isopropyl acetate and tert-butylacetate.
 12. The process according to claim 1, wherein the acid employedis a mineral acid selected from orthophosphoric acid, hydrochloric acidand sulphuric acid.
 13. The process according to claim 1, wherein the pHof the reaction in step (b) is 3.0±0.1.
 14. The process according toclaim 1, wherein the water-miscible organic solvent is selected from aketonic solvent and a nitrile.
 15. The process according to claim 1,wherein the water-miscible organic solvent is a nitrile selected fromacetonitrile, propionitrile and butyronitrile.
 16. The process accordingto claim 1, wherein the water immiscible solvent is selected fromdichloromethane, dichloroethane, chloroform, ethyl acetate, n-butylacetate and isopropyl acetate.
 17. The process according to claim 1,wherein the alkali or an alkaline earth metal containing salt isselected from sodium chloride, potassium chloride, sodium sulphate,potassium sulphate and calcium chloride.
 18. The process according toclaim 1, wherein the anti-solvent is selected from an aromatichydrocarbon and an aliphatic hydrocarbon.
 19. The process according toclaim 18, wherein the aromatic hydrocarbon is selected from toluene andxylene, and the aliphatic hydrocarbon is selected from cyclohexane,n-hexane and heptane.